Restenosis inhibitors after angioplasty, and stents or balloons used to inhibit restenosis after angioplasty.

Abstract

To provide an agent that suppresses the proliferation of vascular smooth muscle cells and can inhibit restenosis, particularly restenosis after stent treatment.SOLUTION: A post-angioplasty restenosis inhibitor is prepared that contains dantrolene or a pharmaceutically acceptable salt thereof or a hydrate thereof as an active ingredient. Preferably, the pharmaceutically acceptable salt is sodium salt. Additionally, an agent-elution stent or an agent-elution balloon is prepared, with its surface coated with an agent layer that contains dantrolene or a pharmaceutically acceptable salt thereof or a hydrate thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a restenosis inhibitor after angioplasty, a stent or balloon used for inhibiting restenosis after angioplasty, and a coating agent for coating a stent or balloon. [Background Art]

[0002] Vascular smooth muscle cells are cells that play an important role in atherosclerosis. Originally, vascular smooth muscle cells function to repair damaged sites by changing their phenotype from a differentiated type (contractile type) to a dedifferentiated type (proliferative type) in response to arterial injury. However, these vascular smooth muscle cells are known to be involved in vascular restenosis after catheter angioplasty for arterial diseases through proliferation, migration, and extracellular matrix production. This catheter angioplasty is frequently used for the treatment of stenosis of coronary arteries and peripheral arteries. To widen the stenosed site of an artery, a balloon is inserted to expand the blood vessel, and then a stent is placed to keep the vessel dilated. The stent is left indwelling in the body as-is to maintain the vascular morphology. Simple metal stents called Bare Metal Stents that have been used in this treatment method have the advantage that the stent is quickly covered by vascular endothelium. On the other hand, due to the proliferation of vascular smooth muscle cells, there is a high risk of vascular restenosis occurring after the procedure.

[0003] Drug-eluting stents were developed to solve this problem of restenosis. In drug-eluting stents, the effect of the drug coated on the stent surface can inhibit the proliferation of vascular smooth muscle cells. The use of this drug-eluting stent greatly reduces restenotic lesions.

[0004] On the other hand, the selection of drugs used in drug-eluting stents is crucial. Drugs used in stents include the immunosuppressant sirolimus and the anticancer drug paclitaxel (see Non-Patent Literature 1). However, if the drug's effect is too strong, it can suppress the proliferation of vascular endothelial cells as well as vascular smooth muscle cells. As a result, stent endothelialization is delayed, leaving the metal stent struts continuously exposed to the blood, increasing the risk of stent thrombosis. Therefore, there has been a need for new drugs that can suppress the proliferation of smooth muscle cells.

[0005] By the way, dantrolene is a compound belonging to the hydantoin derivatives, and it blocks the transmission of excitation from the transverse tubules to the sarcoplasmic reticulum by blocking ryanodine receptors, thereby preventing Ca from the sarcoplasmic reticulum. 2+ It is known to suppress the release of dantrolene. In addition to being widely used as a muscle relaxant, dantrolene is also known to have an inhibitory effect on hepatic fibrosis as shown in Patent Document 1, an inhibitory effect on Ras activity or an inhibitory effect on the proliferation of cancer cells as shown in Patent Document 2, and an effect on spasms after neuropathy as shown in Patent Document 3. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2020-7237 [Patent Document 2] International Publication No. 2015 / 182625 Brochure [Patent Document 3] Japanese Patent Publication No. 2016-539167 [Non-patent literature]

[0007] [Non-Patent Document 1] Kiyoo Ishiwata, Coronary Intervention in the New Era, Biomedical Engineering 43(1):36-42, 2005. [Overview of the project] [Problems that the invention aims to solve]

[0008] The object of the present invention is to provide a drug that can suppress the proliferation of vascular smooth muscle cells and inhibit vascular restenosis after angioplasty, particularly after stent treatment. [Means for solving the problem]

[0009] In order to solve the above problems, the inventors of this invention diligently investigated and focused on dantrolene, which has muscle relaxant effects, as a drug to suppress stenosis. Surprisingly, they found that dantrolene inhibits the proliferation and migration of aortic smooth muscle cells (MOVAS) induced by platelet-derived growth factor (PDGF). Furthermore, dantrolene inhibited the switching of the MOVAS phenotype from contractile to proliferative in both in vitro and in vivo. In addition, they found that dantrolene has an inhibitory effect on neointima formation after arterial injury, thus completing the present invention.

[0010] In other words, the present invention is as follows: [1] A restenosis inhibitor after angioplasty, comprising dantrolene or a pharmaceutically acceptable salt thereof or a hydrate thereof as the active ingredient. [2] The restenosis inhibitor after angioplasty described in [1] above, characterized in that the pharmaceutically acceptable salt is a sodium salt. [3] A drug-eluting stent or drug-eluting balloon whose surface is coated with a drug layer containing dantrolene or a pharmaceutically acceptable salt thereof or a hydrate thereof. [4] A drug-eluting stent or drug-eluting balloon as described in [3] above, used to suppress restenosis after angioplasty. [5] A coating agent for coating stents or balloons, comprising dantrolene or a pharmaceutically acceptable salt thereof or a hydrate thereof as an active ingredient. [Effects of the Invention]

[0011] The restenosis inhibitor for angioplasty of the present invention makes it possible to suppress restenosis after angioplasty. Furthermore, the drug-eluting stent or drug-eluting balloon of the present invention makes it possible to suppress restenosis after angioplasty. [Brief explanation of the drawing]

[0012] [Figure 1A] This figure shows the cell images after 24, 48, and 72 hours of culture in MOVAS cells administered with PDGF and / or dantrolene (DAN) in Example 1. [Figure 1B] This figure shows the relative cell numbers (Relative Cell Numbers % to the 24hr control (DAN(-))) after 24, 48, and 72 hours of culture following administration of PDGF and / or dantrolene (DAN) to MOVAS cells in Example 1, with the cell number after 24 hours of culture being 100% compared to the cell number after 24 hours of culture when only DAN(-) was added. [Figure 2A] This figure shows the cell images after 4, 8, and 12 hours of culture following the administration of PDGF and / or dantrolene (DAN) to MOVAS cells seeded in Culture-Insert 2Wells in Example 2, and then removing the Culture-Insert 2Wells to form a scratch. [Figure 2B] This figure shows the percentage of scratch regions that were not repaired by migration after 4, 8, and 12 hours of culture, following the administration of PDGF and / or dantrolene (DAN) to MOVAS cells seeded in Culture-Insert 2Wells in Example 2, followed by removal of the Culture-Insert 2Wells to form scratches, and subsequent culture. [Figure 3A] This figure shows the immunofluorescence staining image when MOVAS cells were administered PDGF and / or dantrolene in Example 3. [Figure 3B]In Example 3, the figure shows the positive cell rate (%) of SMemb positive cells or the positive cell rate (%) of Calponin-1 positive cells when PDGF and / or dantrolene is administered to MOVAS cells. [Figure 4A] In Example 4, the figure shows the results of hematoxylin-eosin (HE) staining, elastica van Gieson (EVG) staining, and SMemb immunostaining when a guide wire (GW) is inserted into a femoral artery injury model and dantrolene is further applied around the femoral artery. [Figure 4B] In Example 4, the figure shows the calculation results of the SMemb positive cell rate in the intimal area (Positive cell rate(%)) and the lumen area ratio to the total vascular cross-sectional area (Lumen Area / CSA(%)) when a guide wire (GW) is inserted into a femoral artery injury model and dantrolene is further applied around the femoral artery. Mode for Carrying Out the Invention

[0013] <Terminology> 1. Dantrolene Dantrolene (Dantrolene: 1-[[[5-(4-Nitrophenyl)-2-furanyl]methylene]amino]-2,4-imidazolidinedione) has the molecular formula C 14 H 10 N4O5, with a molecular weight of 314.257 and CAS number 7261-97-4, is a compound represented by the following chemical formula (I). Dantrolene can be produced by a known method, and a commercially available compound can also be used.

[0014] Chemical formula

[0015] In this specification, "pharmaceutically acceptable salts" in dantrolene or its pharmaceutically acceptable salts include, for example, alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, transition metal salts such as ammonium salt and zinc salt, cyclic amine salts, mono-, di-, or tri-lower alkylamine salts, mono-, di-, or trihydroxy-lower alkylamine salts, polyhydroxy-lower alkylamine salts, and hydroxy-lower alkyl-lower alkylamine salts, with sodium salt being a preferred example.

[0016] Furthermore, dantrolene or its pharmaceutically acceptable salts may also be solvates thereof with water or alcohol, for example, dantrolene sodium (1-[[[5-(4-Nitrophenyl)-2-furyl]methylene]amino]-3-sodio-2,4-imidazolidinedione) hydrate. Such dantrolene sodium hydrate is commercially available under the trade name "Dantrium®". The above-mentioned Dantorium blocks ryanodine receptors and thus blocks the transmission of excitation from transverse tubules to the sarcoplasmic reticulum, thereby reducing Ca from the sarcoplasmic reticulum. 2+ It is used as a muscle relaxant to suppress the release of [unclear].

[0017] 2. Restenosis after angioplasty In this specification, restenosis after angioplasty includes restenosis of vessels after angioplasty using stents, rotablators, directional coronary atherectomy (DCA), and laser angioplasty, as well as intimal thickening and neointimal proliferation. Sites of restenosis include the coronary arteries, iliac arteries, femoral arteries, popliteal arteries, and tibial arteries.

[0018] 3. Suppression of restenosis after angioplasty In this specification, suppression of restenosis after angioplasty means preventing or reducing restenosis after angioplasty.

[0019] <An agent to suppress restenosis after angioplasty>

[0020] The restenosis inhibitor after angioplasty according to the present invention (hereinafter also referred to as "the restenosis inhibitor") may be administered via a variety of routes, including arteries, veins, muscles, orally, percutaneously, and subcutaneously. Alternatively, the restenosis inhibitor may be directly applied, injected, or sprayed onto the site where angioplasty was performed.

[0021] The restenosis inhibitor in question may further comprise carriers, excipients, and diluents that are commonly used in the manufacture of pharmaceuticals or pharmaceutical compositions. Examples of such carriers, excipients, and diluents include mannitol, polyvinyl alcohol, lactose, dextrose, sucrose, sorbitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Each of these restenosis inhibitors may be used in conventional ways, such as injectable formulations, topical formulations, sprays, or in dosage forms such as powders, granules, tablets, pills, capsules, solutions, oils, suspensions, emulsions, and syrups.

[0022] Furthermore, dantrolene or a pharmaceutically acceptable salt thereof may be encapsulated or embedded in inert polymer particles such as microcapsules, nanocapsules, nanoparticle nanospheres, microspheres, or fine particles.

[0023] The preferred dosage of this restenosis inhibitor can be appropriately determined by those skilled in the art, taking into account various relevant factors such as the patient's age, sex and weight, health status, and disease severity. Specifically, in the case of intravenous administration, when administered to an adult (60 kg) as a restenosis inhibitor after angioplasty, the daily dose of the active ingredient is in the range of 0.01 to 0.5 g, preferably 0.1 to 0.2 g, and can be administered once or in several divided doses. Possible administration intervals include every 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, every 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, or every 30 minutes.

[0024] As embodiments of the restenosis inhibitor in this case, it may be used to prevent restenosis before it occurs, or to suppress the progression of restenosis once it has already occurred.

[0025] The restenosis inhibitor in question can also be provided as a single-agent preparation, along with a package insert stating that it suppresses restenosis after angioplasty by administering dantrolene or a pharmaceutically acceptable salt thereof.

[0026] <Drug-eluting stent or drug-eluting balloon> The drug-eluting stent or drug-eluting balloon according to the present invention (hereinafter also referred to as "the drug-eluting stent or drug-eluting balloon") has a drug layer coated on its surface containing dantrolene or a pharmaceutically acceptable salt thereof. One aspect of the use of this drug-eluting stent or drug-eluting balloon is as follows.

[0027] First, a catheter inserted through an artery is used to guide the drug-eluting stent and / or drug-eluting balloon to the site of vascular stenosis. Next, the drug-eluting stent or balloon is expanded until it is in contact with the site of vascular stenosis. If a stent is used, the stent is left in place. As a result, the site of vascular stenosis widens the vessel, and dantrolene or a pharmaceutically acceptable salt thereof contained in the drug layer is eluted at the site of vascular stenosis, thereby suppressing restenosis.

[0028] The "drug-eluting stent" in this drug-eluting stent or drug-eluting balloon is not particularly limited as long as it is a stent that can appropriately expand the narrowed part of a blood vessel while reducing vascular recoil and eluting a drug. In terms of shape, it may be tubular or mesh. Examples of materials for the stent in this drug-eluting stent include metals such as stainless steel, tantalum, platinum, nickel, cobalt-chromium alloy, and titanium alloy, as well as biodegradable polymers such as polylactic acid, polyglycolic acid, and polyamide, and non-biodegradable polymers such as polyethylene, polyurethane, polyvinyl chloride, polytetrafluoroethylene, and silicon. Furthermore, it is preferable that the material of the stent is a material that satisfies the conditions of biocompatibility.

[0029] The "drug-eluting balloon" in this drug-eluting stent or drug-eluting balloon is not particularly limited as long as it is a balloon that can appropriately expand the narrowed area of ​​a blood vessel and release a drug. Examples of materials for this drug-eluting balloon include polyamide, polyester, polyurethane, or elastomers thereof.

[0030] There are no particular limitations on the method for producing the drug-eluting stent or drug-eluting balloon, but for example, it can be produced by steps (a), (b), and (c), which are: (a) preparing a drug solution containing dantrolene or a pharmaceutically acceptable salt thereof; (b) immersing the stent or balloon in the drug solution to coat the surface of the stent or balloon with the drug solution; and (c) drying the stent or balloon obtained in step (b), which has the drug solution coated on its surface. Alternatively, it can be produced by steps (a), (b-1), and (c), which are: (a) preparing a drug solution containing dantrolene or a pharmaceutically acceptable salt thereof; (b-1) applying or spraying the drug solution onto the stent or balloon to coat the surface of the stent or balloon with the drug solution; and (c) drying the stent or balloon obtained in step (b-1), which has the drug solution coated on its surface. In addition, the method described in Japanese Patent Application Publication No. 2019-81805 is incorporated into the present invention by reference.

[0031] The above-mentioned drug solution can be prepared as appropriate, taking into consideration its adhesion to the stent or balloon, its affinity for dantrolene or a pharmaceutically acceptable salt thereof, and the release rate of dantrolene or a pharmaceutically acceptable salt thereof, but may also contain linear hydrocarbons such as lipolipid alcohols, lipolipid aldehydes, fatty acids, or mixtures thereof; polymers copolymerized by one or more monomers such as methacrylic acid, butyl methacrylate, vinyl acetate, urethane, glycolic acid, lactic acid, or caplocton; or surfactants.

[0032] The amount of dantrolene or a pharmaceutically acceptable salt thereof contained in the above drug solution can be, for example, 0.1 to 100% by weight.

[0033] <Coating agent for coating stents or balloons> The coating agent for coating a stent or balloon according to the present invention (hereinafter also referred to as "the coating agent") comprises dantrolene or a pharmaceutically acceptable salt thereof or a hydrate thereof as an active ingredient, and is a coating agent for coating the surface of a stent or balloon with dantrolene or a pharmaceutically acceptable salt thereof or a hydrate thereof, and the stent or balloon is preferably a stent or balloon used to suppress vascular stenosis.

[0034] The pharmaceutical solution in this coating agent is a pharmaceutical solution containing dantrolene or a pharmaceutically acceptable salt thereof or a hydrate thereof, and the pharmaceutical solution may contain excipients as appropriate, taking into consideration the adhesion or affinity between dantrolene or a pharmaceutically acceptable salt thereof or a hydrate thereof and the stent or balloon, and the release rate of dantrolene or a pharmaceutically acceptable salt thereof. Examples of such excipients include linear hydrocarbons such as lipolipid alcohols, lipolipid aldehydes, fatty acids, or mixtures thereof, or surfactants.

[0035] A drug-eluting stent or balloon can be manufactured by immersing a stent or balloon in the coating agent, or by applying or spraying the coating agent onto a stent or balloon. The material of the stent or balloon in the coating agent is the same as that of the stent or balloon described in the drug-eluting stent or balloon description.

[0036] <Methods to suppress restenosis after angioplasty> Another aspect 1 of the present invention is a method for suppressing restenosis after angioplasty, which includes the step of administering the restenosis inhibitor for angioplasty to a patient who requires suppression of restenosis after angioplasty. The administration route, method of administration, and dosage are as described in the section on the restenosis inhibitor for angioplasty. Another aspect 2 of the present invention is a method for suppressing restenosis after angioplasty, which includes the step of placing the drug-eluting stent or drug-eluting balloon at the stenotic site where angioplasty was performed. [Examples]

[0037] The present invention will be described more specifically below with reference to examples, but the technical scope of the present invention is limited to these The examples are not exhaustive. The statistical analyses used in the examples are as follows: The unpaired t-test was used to statistically compare data obtained in two different situations with normal distributions. The Mann-Whitney test was used to statistically compare two sets of data with non-normal distributions. Furthermore, for normal distributions, one-way analysis of variance (ANOVA) and Tukey's multiple comparison test were used to statistically compare three independent groups. One-way analysis of variance (ANOVA) and Dunnett's multiple comparison test were used to compare the control group with each concentration group. The Kruskal-Wallis (KW) test and Dunn's multiple comparison test were used to statistically compare three independent groups with non-normal distributions.

[0038] [Example 1] Inhibitory effect of platelet-derived growth factor induction on the proliferation of vascular smooth muscle cells First, we investigated whether dantrolene inhibits platelet-derived growth factor (PDGF)-induced proliferation of vascular smooth muscle cells (MOVAS) in vitro.

[0039] Mouse aortic smooth muscle cells (MOVAS cells, ATCC CRL-2797) were seeded at 5,000-6,000 cells / well in 96-well plates and divided into a control group (without PDGF) and a PDGF group. Dimethyl sulfoxide (DMEM: Fujifilm Wako Pure Chemical Industries) supplemented with 10% FBS (Cytiva HyClone) and 1% penicillin-streptomycin (Fujifilm Wako Pure Chemical Industries) was used as the cell culture medium. The PDGF group was given PDGF (20 ng / mL) along with DMSO alone (DAN(-)) or dantrolene sodium (355-44503: Fujifilm Wako Pure Chemical Industries) (DAN 10 μM) dissolved in DMSO. The control group without PDGF was given DMSO alone (DAN(-)) or dantrolene (DAN 10 μM) dissolved in DMSO. Cell counts were measured after 24, 48, and 72 hours of culture using Cell Counting kit-8 (Dojin Chemical Laboratories). Figure 1A shows the cell images after each culture time, and Figure 1B shows the cell count results. The vertical axis in Figure 1B represents the relative cell count to the control group (where only DAN(-) was added after 24 hours of culture) which is set to 100%. Each dot represents a value measured from one well, summarizing the data from the four experiments. *** The Mann-Whitney test was used to compare DAN(-) and PDGF-DAN(-) when P < 0.001 (under unequal variance). ### P <0.001, ## P <0.01, # P <0.05 Unpaired t-test was used to compare DAN(-) and DAN 10μM (assuming equal variances).

[0040] First, as shown in Figures 1A and 1B, PDGF had a strong effect on cell proliferation. Addition of PDGF induced cell proliferation, and a statistically significant difference (P < 0.001) was observed between the control group's DAN(-) and the PDGF group's DAN(-) at 24, 48, and 72 hours. Next, regarding the effect of dantrolene administration, in the PDGF group, the addition of DAN(10 μM) significantly suppressed cell proliferation at 24, 48, and 72 hours compared to the addition of DAN(-). Therefore, it was confirmed that dantrolene inhibits the proliferation of vascular smooth muscle cells (MOVAS).

[0041] [Example 2] Inhibitory effect of platelet-derived growth factor induction on vascular smooth muscle cell migration Next, we investigated whether dantrolene inhibits platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell (MOVAS) migration in vitro using a cell wound healing assay.

[0042] MOVAS cells were seeded in a μ-dish, Culture-Insert 2Well (ibidi). The culture medium used was dimethyl sulfoxide (DMEM: Fujifilm Wako Pure Chemical Industries) supplemented with 10% FBS (Cytiva HyClone) and 1% penicillin-streptomycin (Fujifilm Wako Pure Chemical Industries). This dish is designed to create a uniform intercellular space (scratch) in the cell wound healing assay. When 90% confluence was reached, the medium was replaced with DMEM supplemented with 0.1% FBS. The Culture-Insert 2Well was removed to form a scratch, and the PDGF group was treated by adding PDGF (20 ng / mL) to the medium along with DMSO alone (DAN(-)) or dantrolene sodium dissolved in DMSO (DAN 10 μM: Fujifilm Wako Pure Chemical Industries). The control group without PDGF was treated with either DMSO only (DAN(-)) or dantrolene dissolved in DMSO (DAN 10 μM) in the culture medium. The repair area of ​​the scratched region was measured at 4, 8, and 12 hours. Figure 2A shows the cell images after scratch formation and at each time point, and Figure 2B shows a summary of the results for the scratched region that was not repaired by migration. In Figure 2B, the vertical axis represents the area ratio to the value with the scratched region area at 0h set to 100% under each condition. Each dot represents a value measured from one field of view, and the data from the three experiments are summarized. ### Unpaired t-test was used to compare DAN(-) and PDGF-DAN(-) (assuming equal variances) with P < 0.001. *** The Mann-Whitney test was used to compare DAN(-) and PDGF-DAN(-) when P < 0.001 (under unequal variance). ### P <0.001, ## P <0.01, # P <0.05 Unpaired t-test was used to compare DAN(-) and DAN 10μM (assuming equal variances).

[0043] Figures 2A and 2B show that MOVAS cell migration was significantly activated in the PDGF group compared to the control group (no PDGF treatment). Furthermore, dantrolene administration suppressed the migration of MOVAS cells to the scratch area in both the control and PDGF groups as the culture time increased. Therefore, it was confirmed that dantrolene has an effect of suppressing the migration of vascular smooth muscle cells. Suppressing the migration of vascular smooth muscle cells helps prevent them from migrating beyond the internal elastic lamina and narrowing the lumen.

[0044] [Example 3] Inhibitory effect of PDGF-induced smooth muscle phenotype switching The effect of dantrolene on the phenotype of vascular smooth muscle cells was evaluated by immunohistochemistry. Specifically, immunofluorescence staining was performed for SMemb, a marker of the dedifferentiated (proliferative) type expressed during vascular smooth muscle cell proliferation, and Calponin-1, a marker of the differentiated (contractile) type expressed under normal conditions. The phenotype of vascular smooth muscle cells stimulated with the growth factor PDGF was then examined.

[0045] MOVAS cells were seeded in the same manner as in Example 1. The PDGF group received PDGF (20 ng / mL) along with DMSO (DAN(-)) or dantrolene sodium dissolved in DMSO (DAN 10 μM: Fujifilm Wako Pure Chemical Industries). The control group without PDGF received DMSO only (DAN(-)) or dantrolene dissolved in DMSO (DAN 10 μM). After adding PDGF or DMSO, MOVAS cells were cultured for 48 hours, then fixed with 4% paraformaldehyde for 5 minutes. Permeabilization was performed with 0.1% Triton-X for 20 minutes, and blocking was performed for 60 minutes using 1% bovine serum albumin (BSA) and Protein block (Dako). The primary antibody used was anti-SMemb antibody (YAMASA) or anti-Calponin-1 antibody (D8L2T, Cell Signaling Technology), and the cells were left to stand overnight at 4°C. Cells were labeled with Alexa488-labeled goat anti-mouse secondary antibody or Alexa488-labeled goat anti-rabbit secondary antibody and observed. Immunofluorescence staining images of each cell are shown in Figure 3A, and the percentage (%) of SMemb-positive or Calponin-1-positive cells relative to the total number of cells is shown in Figure 3B. In Figure 3B, each dot represents the percentage of positive cells based on photographs of 3 to 13 fields obtained in a single experiment. ### P < 0.001, ## P < 0.01 An unpaired t-test was used to compare DAN(-) and PDGF-DAN(-) (assuming equal variances). ## Unpaired t-test was used to compare DAN(-) and DAN 10μM (assuming equal variances) with P < 0.01.

[0046] As shown in Figure 3A, in the PDGF group, the shape of MOVAS cells changed to a more elongated, curved crescent shape after the addition of PDGF. This crescent shape is characteristic of dedifferentiated (proliferating) cells. On the other hand, in the control group, which did not receive PDGF stimulation, MOVAS cells were not stained with the proliferation marker SMemb, regardless of dantrolene administration.

[0047] Furthermore, as shown in Figure 3B (left), the expression of SMemb, a proliferation marker, was significantly improved in the DAN(-) group of the PDGF group compared to the control group, showing a positive cell rate of approximately 70%. Therefore, it was revealed that the addition of PDGF induces SMemb expression. On the other hand, the addition of dantrolene in the PDGF group decreased the SMemb positive cell rate. Therefore, it was revealed that dantrolene has the effect of suppressing the switch of the vascular smooth muscle cell phenotype to the dedifferentiated (proliferative) type.

[0048] Furthermore, as shown in Figure 3B (right), the expression of Calponin-1, a contractile marker, was significantly lower in the DAN(-) group of the PDGF group compared to the control group, with a positive cell rate of 4% or less. Therefore, it was revealed that the addition of PDGF suppresses Calponin-1 expression. On the other hand, in the PDGF group, the addition of dantrolene maintained a Calponin-1 positive cell rate at almost the same expression level as in the group without PDGF. Therefore, it was considered that dantrolene has an inhibitory effect on restenosis.

[0049] [Example 4] Inhibitory effect on phenotypic switching of vascular smooth muscle cells after intima-scraping of the femoral artery in mouse We investigated the phenotypic switching of vascular smooth muscle cells using a femoral artery intimal scraping model.

[0050] Using a guidewire (GW), a mouse femoral artery abrasion injury model was created. Four weeks later, the femoral arteries of mice coated with dantrolene sodium mixed with polyvinyl alcohol (PVA) and mice coated with PVA alone were examined, and histological analysis was performed to investigate smooth muscle cell proliferation and phenotypic switching in vivo. Polyvinyl alcohol was used to adhere dantrolene to the blood vessels.

[0051] (Creation of a femoral artery intima scraping model) A femoral artery injury model was created based on the method of Wang et al. (PLoS One. 2014 Feb 27;9(2):e90146. doi: 10.1371 / journal.pone.0090146). Mice were anesthetized and intubated using pentobarbital sodium (70 mg / kg weight, ip), and placed supine under a microscope. After incising the skin above the femoral artery, the femoral nerve was dissected and pulled to the side. The femoral artery was held in place with a loop-shaped 6.0 silk surgical suture for temporary hemostasis, and arterial spasm was prevented by using 1-2 drops of 1% lidocaine. Using micro-scissors, arterial incision was made through a muscular branch. A guidewire (GW) with a diameter of 0.014 or 0.018 G was inserted into the femoral artery through this muscular branch and left in the artery for 1 minute. After removing the GW (gum bandage), the muscular branches were ligated using 4.0 silk sutures. Blood flow in the femoral artery was restored by releasing the sutures. Dantrolene sodium (10 mM: Fujifilm Wako Pure Chemical Industries) was mixed with an equal volume of polyvinyl alcohol (PVA) and applied around the femoral artery to create the GW + DAN group. On the other hand, only PVA was applied without DAN to create the GW + PVA group. The skin was sutured using 6.0 silk sutures. In the control group, the femoral artery was exposed and only the muscular branches were ligated.

[0052] (Histological analysis) Four weeks after the creation of the injury model, the femoral artery injured with a guidewire was fixed with 10% formalin. At least three different levels of sections were selected for morphometric analysis. The obtained sections were stained with hematoxylin-eosin (HE) and Elastica wan-Gieson (EVG). Unstained sections were used for immunofluorescence staining. SMemb immunostaining was also performed using the same method as in Example 3. Figure 4A shows the hematoxylin-eosin (HE) stained, Elastica wan-Gieson stained, and SMemb immunostained images of the femoral artery of GW-injured mice four weeks after injury. Furthermore, Figure 4B shows the calculated SMemb-positive cell rate in the intima area and the ratio of lumen area to total vessel cross-sectional area (Lumen Area / CSA (%)).

[0053] HE staining and EVG staining in Figure 4A revealed that, compared to the control group, the GW+PVA group showed significant intimal proliferation and neointima formation due to the migration and proliferation of smooth muscle cells beyond the internal elastic lamina, resulting in a narrowing of the lumen. Furthermore, Figure 4B showed that numerous SMemb-positive cells were observed in the proliferated intima by immunohistochemistry. On the other hand, the proliferated intima coated with dantrolene showed a low SMemb positivity not only at the dantrolene-coated site but also throughout the surrounding tissue. Moreover, dantrolene coating maintained a lumen area at approximately the same level as the control group. Therefore, it was revealed that dantrolene has an effect of suppressing restenosis and maintaining the phenotype of smooth muscle cells in a differentiated state.

[0054] Conventionally, drugs used in drug-eluting stents have been those that suppress cell division and proliferation, such as anticancer drugs. This has led to the problem of excessive endothelialization, leaving the stent struts exposed to blood flow for extended periods. On the other hand, the above example shows that using dantrolene, known as an inhibitor of the Ca2+ channel RyR2, maintains the differentiated phenotype of vascular smooth muscle and suppresses the proliferation and invasion of vascular smooth muscle. Therefore, using dantrolene makes it possible to suppress vascular stenosis, especially restenosis after angioplasty.

Claims

1. A drug-eluting stent or drug-eluting balloon for inhibiting the proliferation of vascular smooth muscle cells, wherein the surface is coated with a drug layer containing dantrolene or a pharmaceutically acceptable salt thereof or a hydrate thereof, characterized in that the drug-eluting stent or drug-eluting balloon is composed of at least one biocompatible material selected from the group consisting of stainless steel, cobalt-chromium alloy, titanium alloy, polylactic acid, polyglycolic acid, polyamide, polyethylene, polyurethane, polyvinyl chloride, polytetrafluoroethylene, and silicon.

2. A drug-eluting stent or drug-eluting balloon according to claim 1, characterized in that it is composed of at least one biocompatible material selected from the group consisting of polylactic acid, polyglycolic acid, polyamide, polyethylene, polyurethane, polyvinyl chloride, polytetrafluoroethylene, and silicon.

3. A drug-eluting stent or drug-eluting balloon according to claim 1, characterized in that it is composed solely of a biocompatible material selected from the group consisting of stainless steel, cobalt-chromium alloy, and titanium alloy.

Images